Summary Following vascular injury, blood loss is controlled by the mechanisms of hemostasis. During this process, the serine proteinase, thrombin, is generated both locally and rapidly at sites of vessel damage. It plays a pivotal role in clot promotion and inhibition, and cell signaling, as well as additional processes that influence fibrinolysis and inflammation. These functions involve numerous cleavage reactions, which must be tightly coordinated. Failure to do so can lead to either bleeding or thrombosis. The crystal structures of thrombin, in combination with biochemical analyses of thrombin mutants, have provided insight into the ways in which thrombin functions, and how its different activities are modulated. Many of the interactions of thrombin are facilitated by exosites on its surface that bind to its substrates and/or cofactors. The use of cofactors not only extends the range of thrombin specificity, but also enhances its catalytic efficiency for different substrates. This explains a paradox (i.e. thrombin is a specific proteinase, and yet one that has multiple, and sometimes opposing, substrate reactions). In this review, we describe the context in which thrombin acts during hemostasis and explain the roles that its exosites and cofactors play in directing thrombin function. Thereafter, we develop the concept of cofactor competition as a means by which the activities of thrombin are controlled.

IntroductionVon Willebrand factor (VWF) binds exposed collagen to form a bridge between the site of vascular injury and platelets during the initiation of hemostasis. 1,2 Due to its pivotal role in hemostatic plug formation, VWF function may directly influence the likelihood of a thrombotic event, as suggested by the association of VWF levels with an increased risk of ischemic heart disease (odds ratio [OR], approximately 1.5). [3][4][5][6][7][8] Plasma VWF is comprised of multimers held together by intermolecular disulfide bonds. The molecular weight (MW)/ multimeric composition of VWF is a key determinant of its platelet-tethering function. 9,10 Larger multimers are the most reactive at the site of vessel injury. VWF multimeric size is modulated by ADAMTS13, 11-14 which cleaves in the VWF A2 domain, thus reducing both its MW and platelet-tethering function. ADAMTS13 deficiency leads to VWF-induced platelet aggregation, resulting in thrombotic thrombocytopenia purpura (TTP). 15 The level of ADAMTS13 in the blood may thus influence cardiovascular disease. Herein, we report on the relationships between ADAMTS13, VWF, and MI in a large population-based casecontrol study (Study of Myocardial Infarctions Leiden [SMILE]). 16 Patients, materials, and methods SubjectsWe used plasma samples from the previously published population-based case-control study SMILE. We included 560 men (18-70 years), consecutively diagnosed with a first episode of myocardial infarction [MI], between 1994 and 1997, and 646 men without MI who had not received anticoagulants for more than 6 months. The control group was frequency matched to cases on 10-year age groups. Venous blood, anticoagulated using trisodium citrate (1:9 vol/vol), was collected at least 6 months after MI (median, 2.6 years). 16 Aliquots of plasma samples were prepared and stored at Ϫ80°C. Approval for these studies was obtained from the institutional review boards of both the university and general hospitals in Leiden, The Netherlands. Informed consent was provided in accordance with the Declaration of Helsinki. ELISAs for ADAMTS13, VWF, and C-reactive proteinThe ADAMTS13 enzyme-linked immunosorbent assay (ELISA) used polyclonal antibodies raised in rabbits immunized with recombinant ADAMTS13 (rADAMTS13). 17,18 Anti-ADAMTS13 thrombospondin type 1 repeat domains (2-4) (TSP1(2-4)) antibodies were affinity purified and biotinylated for use as the detection antibody. Anti-ADAMTS13 antibodies that had been fully depleted of anti-TSP1(2-4) IgG (5 g/mL) were used as the capture antibody in 96-well plates. Wells were washed and blocked before the addition of 100 L/well plasma samples (diluted 1:20 in PBS), or plasma standards in duplicate, and incubated overnight at 4°C. Wells were washed, and ADAMTS13 was detected using biotinylated anti-TSR1(2-4) antibodies (0.1 g/mL), followed by a streptavidin-horseradish peroxidase conjugate (Amersham Pharmacia, Uppsala, Sweden). Highly purified rADAMTS13 17,18 and pooled normal plasma (Technoclone, Vienna, Austria) were used in standard cur...

ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca 2؉ ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca 2؉ concentrations, a highaffinity functional ADAMTS13 Ca 2؉ -binding site was suggested with K D(app) of 80 M (؎ 15 M) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca 2؉ -binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca 2؉ dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca 2؉ -binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the K D(app) for Ca 2؉ of ADAMTS13, and for D187A and E212A also reduced the V max to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca 2؉ revealed an approximately 13-fold reduction in specificity constant, k cat /K m , contributed by changes in both K m and k cat . These results were corroborated using plasmapurified VWF as a substrate. Together, our results demonstrate that a major influence of Ca 2؉ upon ADAMTS13 function is mediated through binding to a highaffinity site adjacent to its active site cleft.

Summary. Background: The multimeric size and platelettethering function of von Willebrand factor (VWF) are modulated by the plasma metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13). In vitro ADAMTS-13 is susceptible to proteolytic inactivation by thrombin. Objectives: In this study, we aimed to characterize the inactivation of ADAMTS-13 by thrombin and to assess its physiological significance. Methods and results: By N-terminal sequencing of cleavage products, and by mutagenesis, we identified the principal thrombin cleavage sites in ADAMTS-13 as R257 and R1176. Using a library of 76 thrombin mutants, we highlighted the functional importance of exosite I on thrombin in the proteolysis of ADAMTS-13. Proteolysis of ADAMTS-13 by thrombin caused an 8-fold reduction in its affinity for VWF that contributed to its loss of VWF-cleaving function. Intriguingly, thrombin-cleaved ADAMTS-13 both bound and proteolyzed a short recombinant VWF A2 domain substrate (VWF115) normally. Following activation of coagulation in normal plasma, endogenous ADAMTS-13, but not added ADAMTS-13, appeared resistant to coagulation-induced fragmentation. An estimation of the K m for ADAMTS-13 proteolysis by thrombin was appreciably higher than the physiological concentration of ADAMTS-13. This was corroborated by the comparatively low affinity of ADAMTS-13 for thrombin (K D 95 nM). Conclusions: Together, our data suggest that ADAMTS-13 is protected from rapid proteolytic inactivation by thrombin in normal plasma. Whether this remains the case under pathological situations involving elevated/sustained generation of thrombin remains unclear.